Current View of microRNA Processing

Abstract

Small evolutionarily conserved noncoding RNAs, microRNAs (miRNAs), regulate gene expression either by translational repression or by mRNA degradation in mammals. miRNAs play functional roles in diverse physiological and pathological processes. miRNA processing is accurately regulated through multifarious factors. The canonical miRNA processing pathway consists of four sequential steps: (a) miRNA gene is transcribed into primary miRNA (pri-miRNA) mainly by RNA polymerase II; (b) pri-miRNA is processed into precursor miRNA (pre-miRNA) through microprocessor complex; (c) pre-miRNA is exported from the nucleus to the cytoplasm with the assistance of Exportin 5 (EXP5/XP05) protein; and (d) pre-miRNA is further processed into mature miRNA via Dicer. Emerging evidence has also demonstrated that some miRNAs undergo alternative processing pathways. Dysregulation of miRNA processing is closely related to tumorigenesis. Here, we review the current advances in the knowledge of miRNA processing and briefly discuss its impact on human cancers.

Introduction

MicroRNAs (miRNAs) have been reported to be key players in various biological processes. ¹ They globally regulate gene expression at the posttranscriptional level and participate in multiple physiological and pathological contexts, including immune cell development, as well as tumorigenesis.^2–9 miRNAs directly bind to a complementary sequence in the 3’ untranslated region (3'UTR), leading to either target mRNA degradation or protein translation inhibition.^10–13 The fundamental roles of miRNAs draw attention of scientific researchers. miRNA biogenesis pathways also attract more interests for scientists to better understand how miRNAs are processed.

It is widely recognized that there are four main steps for miRNA processing pathway.^14–20 Through RNA polymerase II, miRNA gene is first transcribed from genomic DNA (intronic, intergenic, or polycistronic loci) into long primary transcript (pri-miRNA). miRNAs can also be produced either from separate transcription units or from a common transcription unit. Second, pri-miRNA is cleaved into 70 nt long precursor miRNA (pre-miRNA). This step is carried out by a microprocessor complex consisting of Drosha and DiGeorge Critical Region 8 (DGCR8). DGCR8 can reportedly bind to double-stranded RNA-binding domain of pri-miRNA by specially recognizing its “UGU” motif. Drosha can bind to the basal “UG” motif and the stem region of hairpin structure in pri-miRNA. ²¹ Third, pre-miRNA is exported from the nucleus to the cytoplasm mediated through Ran GTPase Exportin-5 (EXP5/XP05) complex. Finally, pre-miRNA is processed by Dicer-trans activating response RNA-binding protein (TRBP) complex to generate mature miRNA. After entering into an Argonaute protein (AGO)-containing miRNA-induced silencing complex (miRISC) duplex, pre-miRNA unwinds its duplex and generate the mature miRNA strand and its complementary miRNA^* strand. In most cases, the miRNA^* strand can be degraded in the cytoplasm. However, some miRNA^* strands continue performing functional roles in cells.^22,23

It has been established that miRNA biogenesis can be regulated at multiple levels. More and more new regulators, including smads, p53, p63, p73, NF-KB, and RNA-binding protein (RBP) Lin28, have been emerged in some miRNA processing pathways.^24–28 Dysregulated miRNA processing has been well documented to be associated with human cancers. Here, we have reviewed the current understanding of both canonical and noncanonical miRNA biogenesis pathways in mammals and their potential clinical use in human cancers.

Canonical miRNA Processing Pathways

Fine-tuning of miRNA processing is indispensable to maintain a physiological balance of miRNA levels in mammals. ²⁹ It is accurately controlled at both transcriptional and posttranscriptional levels. ³⁰ Multiple factors can contribute to miRNA processing and/or their functional roles, including the activities of miRNA processing enzymes, as well as the stability of pri-miRNAs and pre-miRNAs. ³¹

Pri-miRNA biogenesis

The first step of miRNA biogenesis is the transcription from genomic DNA largely by RNA polymerase II, and it generates stem-loop structural pri-miRNAs with a stable 7-methylguanosine cap and poly-(A) tail.^32–34 A common set of transcription factors, including Myc, p53, and REST, can either enhance or block some miRNA biogenesis during the transcription step.^35–38 Other studies have shown that platelet-derived growth factor receptor and the activin/transforming growth factor-β, as well as brain-derived neurotrophic factor, contributed to pri-miRNA biogenesis.^39–41 NF-κB can regulate pri-miR-155 transcription as well as the transcriptional activity of let-7a-3.^42,43 It is also noted that the transcription of miR-34, miR-200, miR-15a, and miR-16-1 can be regulated by p53.^44–47 Another study suggested that proto-oncogene Myb can bind to the pri-miR-15a promoter in K562 cells. Reciprocally, miR-15a can regulate the Myb gene transcripts. ⁴⁸ It has been shown that Runx1 regulates miR-27a expression by directly binding to the miR-27a promoter. ⁴⁹ It has been reported that transcription factors NFI-A and C/EBP alpha can bind to the miR-223 promoter. ⁵⁰ A subsequent study has shown that SMAD proteins regulate the miR-21 biogenesis through a sequence-specific mechanism. ⁵¹

Furthermore, RNA editing can influence pri-miRNA levels in the nucleus. Hydrolytic deamination of adenosine (A-I) of pri-miR-142 can be edited by adenosine deaminase acting on RNA (ADAR) and it can also be degraded by a ribonuclease known as Tudor-SN. ⁵²

Processing from pri-miRNA to pre-miRNA

Recent research has demonstrated that pre-miRNAs can be endowed with the stem-loop region of pri-miRNAs and recognized by the EXP5/XPO5 protein. ⁵³ It is well established that multiple signal transduction molecules interact with Drosha, participating in the transition from pri-miRNA to pre-miRNA. ⁵⁴ Under nutrient-rich conditions, targeting rapamycin complex 1 (mTORC1) can induce Drosha degradation by affecting E3 ubiquitin Mdm2 either in a p53-dependent transcription pathway or in a p53-independent posttranscriptional manner, leading to overall miRNA biogenesis reduction.^55,56 Drosha expression can be enhanced in a low-glucose condition. Estrogen receptor alpha (ERα) also inhibits Drosha-mediated pri-miRNA processing. ⁵⁷ Investigators have found that p68 and p72 in the microprocessor complex are important for some miRNA biogenesis. ⁵⁸ Furthermore, p53 can regulate Drosha-mediated miRNA processing by binding to p68. ⁵⁹

Other work has shown that menin (coded by men1 gene) engages in both miR-let-7a and miR-155 maturation from pri-miRNA to pre-miRNA by binding to RBP ARS2. ⁶⁰ In addition to the above description, RBPs Lin28a/b can block pre-let-7 processing by recruiting the uridyltransferase Zcchc11 and by interacting with the stem-loop of pre-let-7.^61,62

Pre-miRNA exporting into cytoplasm by Exportin-5

It has become increasingly clear that EXP5/XPO5 belongs to importin-β family, acting as a major exporter of nuclear RNAs. ⁶³ EXP5/XPO5 is the rate-determining step in the miRNA processing with a low expression in mammals. ⁶⁴ An additional report has described that HASTY (HST) is the homolog of XPO5 in plants. ⁶⁵ It is also worth noting that HST defect can block a general accumulation of miRNAs.^66,67

Mature miRNA generation

Pre-miRNA processing is further assisted by Dicer, producing a ~22-nucleotide miRNA duplex, with one duplex strand called miRNA^* degraded and the other strand binding to the 3'UTR of target mRNA, leading to either mRNA cleavage or translational inhibition (Fig. 1). Interestingly, some miRNA^* can still play functional roles in cells.^22,23

Figure 1

Current view of miRNA processing. miRNA gene is mainly transcribed by RNA polymerase II (RNA pol II) in the nucleus. After transcription, pri-miRNA is generated with a 7-methyguanosine cap and 3’ poly A tail. Pri-miRNA is cropped into 70-100 nt pre-miRNA by Drosha-DGCR8 complex. Pre-miRNA is exported into the cytoplasm with the assistance of the complex consisting of Ran-GTP and Exportin-5. Pre-miRNA is cleaved by Dicer with its partners AGO and TRBP proteins to form a 20-25 nt miRNA: miRNA^* duplex. Subsequent maturation generates the mature miRISC complex with one strand, whereas the other strand (miRNA^*) is usually degraded. Mature miRNA facilitates the target mRNA cleavage and/or translational repression. Specifically, there are some non-classical pathways. First, a non-classical pathway generating pri-miRNA is conducted from a branched miRtron. Second, miRNA is cleaved by AGO protein to form a mature miRNA in a Dicer-independent manner, for instance, pre-miR-451. Third, miRNA can directly bind to RBPs to prevent from binding their RNA targets via a RISC-independent pathway.

The pre-miRNA maturation can also be regulated by Dcl1 in the nucleus of plants. ⁶⁸ TRBP, a major component of Dicer complex, can be regulated by mitogen-activated kinase/extracellular-signal-regulated kinases (MAPK/ERK) signaling pathway, and it can affect some growth-promoting miRNA processing. ⁶⁹ The single-stranded RNA-binding KH-type splicing regulatory protein has been reported as an interacting protein with Dicer, and it can bind to the stem-loop of pre-miRNAs. ⁷⁰ It has been illustrated that the Akt kinase-induced activation on the let-7a-1 processing can be counteracted by the splicing factor hnRNPA1 through binding to the stem-loop region of pre-let-7a. ⁷¹

Other factors altering miRNA processing

It has been appreciated that the reciprocal and double-negative regulatory feedback loop also exists in some miRNAs biogenesis.^72–74 For example, let-7 can bind to the 3'UTR of myc mRNA by interacting with RBP HuR. ⁷⁵ On the other hand, myc can regulate let-7 processing by promoting Lin28, and it can also directly regulate let-7 expression at the transcriptional level.^76,77

Noncanonical miRNA Biogenesis Pathway

Noncanonical miRNA biogenesis pathway is emerging as a highlight in some miRNA biogenesis either by Drosha-independent or by Dicer-independent pathways. Mirtrons, characterized in Drosophila, are a real addition to the canonical miRNA pathway in the nucleus.^78,79 However, the hairpin pre-miRNA still needs the aid from EXP5/XPO5 protein to be exported into cytoplasm and subsequently cleaved by Dicer. ⁸⁰ Furthermore, transfer RNAs (tRNAs) and small nucleolar RNAs (snoRNAs) have alternative miRNA biogenesis pathways independent of Drosha. ⁸¹ Pre-miR-320 is transcribed directly from genome DNA that bypasses Drosha. ⁸² Similarly, Dicer-independent pathways can produce functional miRNAs. ⁸³ The biogenesis of blood-specific miR-451 is in a Dicer-independent manner, but it still requires the assistance from Drosha. ⁸⁴ In Dicer-deficient cells, mature miR-451 can be generated, while the processing of other miRNAs is impaired. ⁸⁵ It is worth noting that miRNAs can bind to RBPs to hinder RBPs from binding to their RNA targets via a RISC-independent pathway. ⁸⁶ Interestingly, recent studies have also implied that long noncoding RNAs can regulate miRNA biogenesis and that the circular RNAs can potentially soak up miRNAs, providing another level of modulating miRNAs.^87–89

miRNA Biogenesis and Cancer

Dysregulation of miRNA biogenesis has been frequently observed in multiple human cancers.^90,91 A global down-regulation of mature miRNAs via miRNA profiling has also been found in some human cancers.^92–95 Processing of some miRNAs, including miR-26b and miR-125b, is impaired in the transition from pri-miRNA to pre-miRNA in human thyroid anaplastic carcinoma. ⁹⁶ miRNA-processing factor defect reportedly occurs in lung cancer and ovarian cancer.^97,98 Alterations of the key factors in miRNA processing can significantly affect cancer initiation and progression as well as tumor metastasis either by genomic mutations or by aberrant expression.

The expression levels of miRNA processing enzymes such as Dicer, Drosha, and EXP5/XPO5 protein have been found to be positively correlated with invasive carcinomas.^99,100 In bladder urothelial carcinoma cells, Dicer and/or Drosha knockdown by siRNAs can decrease tumor cell proliferation and promote tumor cell apoptosis at the posttranscriptional levels.^99–101 Furthermore, inactivated EXP5/XPO5 protein can promote pre-miRNAs retention in the nucleus. EXP5/ XPO5 restoration shows tumor suppressive effects. ¹⁰² It has been reported that Dicer expression is strongly correlated with human cancers. In ovarian cancer, lung cancer, and breast cancer, enhanced Dicer expression is associated with clinical prognosis. But it has opposite effects in colorectal cancer and prostate cancer.^103–106 TRBP knockdown can destabilize Dicer and impair the miRNA processing in cancer cells. ¹⁰⁷ However, the mechanisms of miRNA biogenesis are complex and still remain to be further characterized. Identifying other factors such as RBPs or other noncoding RNAs in the miRNA processing will provide potential strategies for cancer therapy.

Conclusions and Perspectives

Over the past two decades, it has become increasingly clear that investigating the mechanisms of miRNA processing would be critical to decipher complicated regulatory networks mediated by miRNAs in mammals. However, we are still lacking deeper understanding of miRNA machinery and its physiological roles in various biological aspects. Here, we have provided an overview of where and how miRNAs are processed, including the canonical and noncanonical pathways, and how it is linked to human cancers.

We believe that future studies will focus on several major points: (a) to identify more factors that are involved in miRNA processing; (b) to elucidate how other noncoding RNAs such as piRNAs interact with miRNA biogenesis pathways; (c) to characterize the physiological functions of key factors such as Exportin-5 in human diseases; (d) to discover more non-classical pathways in miRNA processing; (e) to research on miRNA processing pathways in other species; and (f) to identify the differences of miRNA processing pathways between a common set of miRNAs and extracellular miRNAs such as exosomal miRNAs.

Herein, we bring together much of the current work regarding miRNA processing. It has so far only touched the surface of this fertile field of research. Thus, knowledge of miRNA processing in the physiological and pathological roles should be advanced in the near future. Further research will undoubtedly result in new discoveries regarding cancer therapy.

Author Contributions

All the authors conceived, organized, drafted, reviewed, and approved the manuscript.